Active immunotherapy against cancer is hindered by immune tolerance to self, as the majority of antigens expressed on tumor cells are in fact normal self antigens. We have investigated using an agonist monoclonal antibody (mAb) against GITR (glucocorticoid-induced tumor necrosis factor (TNF) receptor) to help overcome this barrier. GITR is a TNF receptor family member expressed highly on activated effector CD4+ and CD8+ T cells, as well as on Foxp3+ regulatory T cells (Tregs) which suppress self- (and therefore tumor-) reactivity. Ligating GITR using an agonist mAb can induce or exacerbate autoimmunity in mouse models, demonstrating the capacity for enhanced self-recognition. We have found using a melanoma mouse model that GITR ligation enhances immunity to melanoma self antigens, and can augment both active and passive immunotherapy against a poorly immunogenic tumor, with focal hypopigmentation as the only observable autoimmunity. However, the consequences of in vivo GITR ligation on effector and regulatory T cells in a tumor-bearing host, as well as the cellular subpopulations responsible for the anti-tumor effects, have not been well-defined. We hypothesize that GITR signaling during active immunization directly co-stimulates tumor antigen-specific CD4+ and CD8+ T cells, leading to enhanced proliferation, effector function and survival. In addition, we propose that GITR ligation further improves tumor immunity by disabling Tregs at the tumor site and in draining lymph nodes. The specific aims are to: 1) Characterize the effects of GITR ligation on CD4+ and CD8+ effector T cells; 2) Investigate the effects of GITR ligation on CD4+Foxp3+ Tregs in tumor-bearing hosts; and 3) Determine the lymphocyte subpopulation(s) that anti-GITR mAb targets to generate enhanced tumor immunity, using in vivo transfer of GITR-positive or GITR-deficient cells. [unreadable] [unreadable] These studies are relevant because immune-modulating antibodies (such as anti-CTLA4 antibody) have activity in patients with advanced cancer, and antibodies targeting human GITR are already in development. Gaining greater mechanistic understanding of the effects of anti-GITR antibody on tumor immunity will allow for more rational translation of this promising immunotherapeutic approach to the clinic. [unreadable] [unreadable] [unreadable]